Method of Diagnosis of Infection by Mycobacteria and Reagents Therefor

Abstract

The present invention provides a method of specifically detecting the presence of one or more Mycobacteria of the M. tuberculosis complex, said method comprising detecting ilvC nucleic acid of one or more Mycobacteria of the M. tuberculosis complex in a sample under conditions that do not detect ilvC nucleic acid of the M. avium complex. The invention also provides methods of diagnosis and treatment of tuberculosis in a subject employing the specific detection ilvC nucleic acid of one or more Mycobacteria of the M. tuberculosis complex.

1 . A method of specifically detecting the presence of one or more Mycobacteria of the  M. tuberculosis  complex, said method comprising detecting ilvC nucleic acid of one or more Mycobacteria of the  M. tuberculosis  complex in a sample under conditions that do not detect ilvC nucleic acid of the  M. avium  complex. 
     
     
         2 . The method according to  claim 1 , wherein detecting ilvC nucleic acid of one or more Mycobacteria of the  M. tuberculosis  complex comprises amplifying one or more ilvC nucleic acids to thereby produce amplified ilvC nucleic acid and detecting the amplified nucleic acid, wherein the detection of amplified ilvC nucleic acid is indicative of the presence of one or more of said Mycobacteria of the  M. tuberculosis  complex in the sample. 
     
     
         3 . The method of  claim 1 , wherein the detected ilvC nucleic acid comprises a sequence of  M. tuberculosis  ilvC DNA or RNA. 
     
     
         4 . The method of  claim 1 , wherein the  M. tuberculosis  complex organism is selected from  M. tuberculosis, M. bovis, M. africanum, M. canetti  and  M. microti  or a combination thereof. 
     
     
         5 . The method of  claim 4 , wherein the  M. tuberculosis  complex organism is selected from  M. tuberculosis  and  M. bovis  or a combination thereof. 
     
     
         6 . The method of  claim 4 , wherein the  M. tuberculosis  complex organism is  M. tuberculosis.    
     
     
         7 . The method according to  claim 6 , wherein the  M. tuberculosis  is a clinical strain or clinical isolate of  M. tuberculosis.    
     
     
         8 . The method of  claim 4 , wherein the  M. tuberculosis  complex organism is  M. bovis.    
     
     
         9 . The method according to  claim 1 , wherein a  Mycobacterium  of the  M. avium  complex is  M. avium.    
     
     
         10 . The method according to  claim 1 , wherein a  Mycobacterium  of the  M. avium  complex is  M. intracellulaire.    
     
     
         11 . The method according to  claim 1 , wherein detecting ilvC nucleic acid of one or more Mycobacteria of the  M. tuberculosis  complex comprises performing an amplification reaction with thermal cycling. 
     
     
         12 . The method according to  claim 1 , wherein detecting ilvC nucleic acid of one or more Mycobacteria of the  M. tuberculosis  complex comprises performing an amplification reaction without thermal cycling. 
     
     
         13 . The method according to  claim 11 , wherein the amplification reaction is performed using a single-stranded or double-stranded cDNA template or DNA/RNA hybrid molecule produced by reverse-transcription of RNA. 
     
     
         14 . The method according to  claim 1 , wherein detecting ilvC nucleic acid of one or more Mycobacteria of the  M. tuberculosis  complex without detecting ilvC nucleic acid of the  M. avium  complex comprises detecting ilvC nucleic acid comprising a sequence of at least 20 contiguous nucleotides in length of SEQ ID NO: 2 or a homologous sequence thereto from one or more Mycobacteria of the  M. tuberculosis  complex. 
     
     
         15 . The method according to  claim 14 , comprising detecting ilvC nucleic acid comprising a sequence of at least 40 contiguous nucleotides in length of SEQ ID NO: 2 or a homologous sequence thereto from one or more Mycobacteria of the  M. tuberculosis  complex. 
     
     
         16 . The method according to  claim 14 , comprising detecting ilvC nucleic acid comprising a sequence of at least 50 contiguous nucleotides in length of SEQ ID NO: 2 or a homologous sequence thereto from one or more Mycobacteria of the  M. tuberculosis  complex. 
     
     
         17 . The method according to  claim 1 , wherein detecting ilvC nucleic acid of one or more Mycobacteria of the  M. tuberculosis  complex comprises amplifying one or more ilvC nucleic acids to thereby produce amplified ilvC nucleic acid and detecting the amplified nucleic acid and wherein said amplifying produces an amplicon of at least 50 contiguous nucleotides in length of SEQ ID NO: 2 or a homologous sequence thereto from one or more Mycobacteria of the  M. tuberculosis  complex. 
     
     
         18 . The method according to  claim 17 , wherein the amplicon comprises at least 80 contiguous nucleotides in length of SEQ ID NO: 2 or a homologous sequence thereto from one or more Mycobacteria of the  M. tuberculosis  complex. 
     
     
         19 . The method according to  claim 1 , wherein detecting ilvC nucleic acid of one or more Mycobacteria of the  M. tuberculosis  complex without detecting ilvC nucleic acid of the  M. avium  complex comprises performing an amplification reaction using one or a plurality of primers that each comprise a sequence of at least about 18 contiguous nucleotides of SEQ ID NO: 2 from position 420 to position 600 thereof, or a sequence complementary to SEQ ID NO: 2 from position 420 to position 600 thereof. 
     
     
         20 . The method according to  claim 1 , wherein detecting ilvC nucleic acid of one or more Mycobacteria of the  M. tuberculosis  complex without detecting ilvC nucleic acid of the  M. avium  complex comprises performing an amplification reaction using one or a plurality of primers that each comprise a sequence of at least about 18 contiguous nucleotides of SEQ ID NO: 2 from position 40 to position 180 thereof, or a sequence complementary to SEQ ID NO: 2 from position 40 to position 180 thereof. 
     
     
         21 . The method according to  claim 1 , wherein detecting ilvC nucleic acid of one or more Mycobacteria of the  M. tuberculosis  complex without detecting ilvC nucleic acid of the  M. avium  complex comprises performing an amplification reaction using one or a plurality of primers that each comprise a sequence of at least about 18 contiguous nucleotides of SEQ ID NO: 2 from position 880 to position 1000 thereof, or a sequence complementary to SEQ ID NO: 2 from position 880 to position 1000 thereof. 
     
     
         22 . The method according to  claim 1 , wherein detecting ilvC nucleic acid of one or more Mycobacteria of the  M. tuberculosis  complex without detecting ilvC nucleic acid of the  M. avium  complex comprises performing an amplification reaction for less than 30 amplification cycles. 
     
     
         23 . The method according to  claim 22 , wherein detecting ilvC nucleic acid of one or more Mycobacteria of the  M. tuberculosis  complex without detecting ilvC nucleic acid of the  M. avium  complex comprises performing an amplification reaction for about 12 amplification cycles to about 27 amplification cycles. 
     
     
         24 . The method according to  claim 22 , wherein detecting ilvC nucleic acid of one or more Mycobacteria of the  M. tuberculosis  complex without detecting ilvC nucleic acid of the  M. avium  complex comprises performing an amplification reaction for about 14 amplification cycles to about 20 amplification cycles. 
     
     
         25 . The method according to  claim 1 , wherein detecting ilvC nucleic acid of one or more Mycobacteria of the  M. tuberculosis  complex without detecting ilvC nucleic acid of the  M. avium  complex comprises performing an amplification reaction on less than about 2 ng/ml input prokaryotic nucleic acid. 
     
     
         26 . The method according to  claim 1 , wherein detecting ilvC nucleic acid of one or more Mycobacteria of the  M. tuberculosis  complex without detecting ilvC nucleic acid of the  M. avium  complex comprises performing an amplification reaction using one or a plurality of amplification primers and a labelled probe comprising a sequence capable of hybridizing to a nucleic acid product of the amplification primers to thereby produce a detectable signal. 
     
     
         27 . The method according to  claim 1 , wherein detecting ilvC nucleic acid of one or more Mycobacteria of the  M. tuberculosis  complex without detecting ilvC nucleic acid of the  M. avium  complex comprises performing an amplification reaction using one or a plurality of amplification primers and a labelled probe capable of binding to at least one of the amplification primers. 
     
     
         28 . The method according to  claim 1 , wherein the sample comprises a sample selected from the group consisting of cultured  M. tuberculosis  cells, sputum, broncho alveolar lavage (BAL), a lymph node biopsy, blood, serum, plasma, a fraction of blood, a fraction of serum, a fraction of plasma and urine. 
     
     
         29 - 33 . (canceled) 
     
     
         34 . The method according to  claim 1 , wherein detecting ilvC nucleic acid of one or more Mycobacteria of the  M. tuberculosis  complex without detecting ilvC nucleic acid of the  M. avium  complex comprises performing an amplification reaction under conditions sufficient to detect at least about 10 4  copies of ilvC nucleic acid in less than about 60 minutes. 
     
     
         35 . The method according to  claim 1 , wherein detecting ilvC nucleic acid of one or more Mycobacteria of the  M. tuberculosis  complex without detecting ilvC nucleic acid of the  M. avium  complex comprises performing an amplification reaction under conditions sufficient to detect at least about 10 3  copies of ilvC nucleic acid in less than about 60 minutes. 
     
     
         36 . The method according to  claim 1 , wherein detecting ilvC nucleic acid of one or more Mycobacteria of the  M. tuberculosis  complex without detecting ilvC nucleic acid of the  M. avium  complex comprises performing an amplification reaction under conditions sufficient to detect at least about 10 4  CFU/ml Mycobacteria of the  M. tuberculosis  complex. 
     
     
         37 . The method according to  claim 1  further comprising detecting one or more nucleic acids of one or more Mycobacteria of the  M. tuberculosis  complex in a sample wherein the one or more nucleic acids are other than ilvC nucleic acid. 
     
     
         38 . The method according to  claim 37 , wherein the one or more nucleic acids other than ilvC nucleic acid is 16s rRNA. 
     
     
         39 . The method according to  claim 37 , wherein the one or more nucleic acids other than ilvC nucleic acid encodes a protein selected from the group consisting of BSX, S9, Rv1265, EF-Tu, P5CR, TetR-like protein and glutamine synthetase or nucleic acid complementary thereto. 
     
     
         40 . The method according to  claim 39 , comprising performing polymerase chain reaction (PCR) to thereby detect nucleic acid comprising a sequence of at least 20 contiguous nucleotides in length of a sequence selected from the group consisting of SEQ ID NOs: 4, 6, 8, 10, 12, 14 and 16. 
     
     
         41 . The method according to  claim 37 , comprising performing an amplification reaction to thereby produce an amplicon of at least 50 contiguous nucleotides in length of the one or more nucleic acids other than ilvC nucleic acid when said nucleic acid is present in the sample. 
     
     
         42 . A process for diagnosing an infection by one or more Mycobacteria of the  M. tuberculosis  complex in a subject, said process comprising performing the method according to  claim 1  on a biological sample from a subject to thereby detect ilvC nucleic acid of one or more Mycobacteria of the  M. tuberculosis  complex in a sample under conditions that do not detect ilvC nucleic acid of the  M. avium  complex wherein the detection of one or more ilvC nucleic acids of one or more Mycobacteria of the  M. tuberculosis  complex is indicative of infection. 
     
     
         43 . The process according to  claim 42 , wherein the infection is an active infection. 
     
     
         44 . The process according to  claim 42 , wherein the infection is a latent infection. 
     
     
         45 . A process for diagnosing tuberculosis in a subject, said process comprising performing the method according to  claim 1  on a biological sample from a subject to thereby detect ilvC nucleic acid of one or more Mycobacteria of the  M. tuberculosis  complex in a sample under conditions that do not detect ilvC nucleic acid of the  M. avium  complex wherein the detection of one or more ilvC nucleic acids of one or more Mycobacteria of the  M. tuberculosis  complex is indicative of tuberculosis. 
     
     
         46 . The process according to  claim 45 , wherein the tuberculosis is pulmonary tuberculosis. 
     
     
         47 . The process according to  claim 45 , wherein the tuberculosis is extrapulmonary tuberculosis. 
     
     
         48 . The process according to  claim 45  further comprising diagnosing one or more clinical symptoms of tuberculosis in the subject. 
     
     
         49 . The process according to  claim 45  further comprising diagnosing one or more clinical symptoms of immune suppression in the subject. 
     
     
         50 . The process according to  claim 45  further comprising diagnosing HIV infection in the subject. 
     
     
         51 . A process for treating tuberculosis or infection by one or more mycobacteria of the  M. tuberculosis  complex, said process comprising:
 (i) performing the method according to  claim 1  on a sample from a subject to thereby detect one or more mycobacteria of the  M. tuberculosis  complex in the sample; and   (ii) administering a therapeutically effective amount of a pharmaceutical composition to the subject to thereby reduce the number of pathogenic bacilli in the lung, blood or lymph system of the subject.   
     
     
         52 . The method according to  claim 12 , wherein the amplification reaction is performed using a single-stranded or double-stranded cDNA template or DNA/RNA hybrid molecule produced by reverse-transcription of RNA.